Synthesis of prostaglandin H2 by prostaglandin H synthase (PHS) results in a two-electron oxidation of the enzyme. An active reduced enzyme is regenerated by reducing cofactors, which become oxidized. This report examines the mechanism by which PHS, from ram seminal vesicle microsomes, catalyzes the oxidation of the reducing cofactor N-acetylbenzidine (ABZ). During the conversion of 0.06 mM ABZ to its final end product 4'-nitro-4-acetylamino-biphenyl, a new metabolite was observed when 1 mM ascorbic acid was present with either 0.2 mM arachidonic acid or 0.5 mM H2O2 as substrate. This metabolite co-eluted with synthetic N'-hydroxy-N-acetylbenzidine (N'HA) and not N-hydroxy-N-acetylbenzidine. The new metabolite was identified as N'HA by ESI/MS/MS. N'HA represented as much as 10% of the total radioactivity recovered by HPLC. When N'HA was substituted for ABZ, PHS metabolized N'HA to 4'-nitro-4-acetylaminobiphenyl. Inhibitor studies demonstrated that metabolism was due t o PHS not cytochrome P-450. DMPO, mannitol, and superoxide dismutase did not alter metabolism. Oxygen uptake studies did not demonstrate a requirement for molecular oxygen. When [18O]H2O2 was used as substrate, 18O enrichment was observed for 4'-nitro-4-acetylamino-biphenyl, but not N'HA. A 97% enrichment was observed for one atom of 18O and 17 + 7% for two 18O atoms. The rapid exchange of 18O-N'HA with water was suggested to explain the lack of enrichment of N'HA and the low enrichment of two 18O atoms into 4'-nitro-4-acetylaminobiphenyl. Results demonstrate a peroxy-genase oxidation of ABZ and N'HA by PHS and suggest stepwise oxidation of ABZ to N'-hydroxy, 4'-nitroso, and 4'-nitro products.